Compositions for prevention of inflammation and adhesion formation uses thereof

ABSTRACT

Pharmaceutical compositions comprising hyaluronic acid, non-steroidal inflammatory drugs, and at least one organic salt of at least one divalent metal, and methods of using these compositions for the inhibition of inflammation and adhesion formation are described. Preferred organic salts of divalent metals include magnesium acetate, zinc acetate, calcium gluconate, and ferrous gluconate. A preferred non-steroidal anti-inflammatory drug is cyclosporin A.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/684,653, filed on Jul. 19, 1996 now U.S. Pat. No. 5,866,554.

BACKGROUND OF THE INVENTION

The invention relates to the inhibition or prevention of inflammationand adhesion formation using pharmaceutical compositions containinghyaluronic acid, non-steroidal inflammatory drugs, and at least oneorganic salt of at least one divalent metal selected from the groupconsisting of magnesium acetate, zinc acetate, calcium gluconate, andferrous gluconate.

During the normal healing process in response to tissue trauma, initialdamage to the mesothelial layer evokes an inflammatory response which ismediated by activation of the kinin, complement and clotting systems(Stangel et al., J. Reprod. Med. 29:143, 1984). A serofibrinous exudateincluding kinin, prostaglandins, and histamine is produced, whichincreases the permeability of local blood vessels (Buckman et al., J.Surg. Res. 20:1, 1976; Stangel et al., supra). Leukocytes are attractedto the site by chemotaxis and the fibrinous exudate is attracted to thedamaged tissue surface due to permeability factors. Fibrin is present infree blood as fibrinogen and it is also a normal product of the clottingcascade. During coagulation, thin strands of fibrinous adhesions form onthe damaged surface within the first three hours (Stangel et al. supra).The majority of these fibrinous adhesions are lysed within the first 72hours as part of the normal healing process and never represent any typeof clinical complication for the individual.

In some cases, however, these initial fibrinous adhesions persist andbecome organized into permanent, thick, fibrous adhesions whichrepresent a significant clinical problem across many different fields ofmedicine. This phenomenon is often associated with joint disease,accidental trauma, and surgical procedures, and is thought to betriggered by an adverse response to many common stimuli including, forexample, an overwhelming inflammatory response, tissue ischemia, and theloss of natural physical tissue separation. For example, post-surgicalperitoneal adhesions are one of the leading causes of intestinalblockage or obstruction (Ellis, Surg. Gynecol. Obstet. 133:497, 1971),and are also of great concern to surgeons who attempt to improvefertility in women through reconstruction. Pelvic adhesions can impairfertility by interfering with the ability of the fallopian tubes to pickup the ovum (Holtz, Fertil. Steril. 41:497, 1984; Diamond and Hershlag,Prg. Clin. Biol. Res. 358:23, 1990). The formation of permanentadhesions in tendons and joints is a major cause of decreased mobilityand chronic pain.

Most research on adhesion prevention agents pertains to post-surgicalabdominal adhesion, and minor efforts have been directed to otheradhesions such as those experienced in the thorax and tendons. Some ofthese studies focused on the prevention of fibrin deposition as astrategy to prevent post-surgical adhesion. These strategies includedthe use of anticoagulants (Holtz, Prog. Clin. Biol. Res. 381:81, 1993);and irrigation (Tulandi, Prog. Clin. Biol. Res. 381:149, 1993); and theseparation of tissue surfaces using (a) dextran lavage ofintraperitoneal procedures (diZerega, Prog. Clin. Biol. Res. 381:1,1993); (b) oxidized cellulose films, e.g., Interceed (diZerega, supra);(c) carboxymethyl cellulose solution (Elkins et al., Fertil. Steril.41:929, 1984); (d) chondroitin sulfate solutions (Oelsner, J. Reprod.Med. 32:812, 1987); (e) polyvinyl pyrrolidine solutions (Goldberg etal., Arch. Surg. 115:776, 1980); (f) Polyoxamer 407 solutions(Steinleitner et al., Obstet. Gynecol. 77:48, 1991); and (g) Gortexfilms (Boyers et al., Prog. Clin. Biol. Res. 358:93, 1990).

Other approaches to prevent post-surgical adhesions have relied on the(a) removal of fibrin matrix through the use of fibrinolytics andproteolytic enzymes (Kapur et al., Arch. Surg. 105:761, 1972; Stangel etal. 1984, supra) or tissue-type plasminogen activator (Evans, Am. J.Surg. 165:229, 1993); and (b) surgical removal of adhesions (Stangel etal. 1984, supra).

Still other strategies have focused on inhibiting the overwhelminginflammatory response to injury which has been found to be one of thekey factors in adhesion formation. A variety of pharmacological agentswhich have anti-inflammatory properties, including corticosteroid,antihistamines, antiprostaglandins, and non-steroidal anti-inflammatoryagents have been investigated in terms of their potential asanti-adhesion agents. Of those agents studied, non-steroidalanti-inflammatory drugs (NSAIDs) have been reported to be among the mostsuccessful in inhibiting inflammation and adhesions formation (diZerega,Prog. Clin. Biol. Res. 381:1, 1993). However, early clinical studieswere not as encouraging as expected, based on earlier animal studies.One of the main concerns in administering NSAIDs is the method ofdelivery. Systemic delivery of the drugs requires sufficient bloodsupply to the injured site; however, this is often disrupted duringsurgery. Thus, tissue ischemia not only stimulates adhesion formation italso prevents the effective systematic delivery of NSAIDs (diZerega,1993 supra). Continuous delivery of the NSAID, tolmetin sodium, wasreported to be highly effective in preventing peritoneal adhesions(Rodgers et al., Int. J. Fertil. 35:40, 1990). However, continuous drugdelivery is impractical in a clinical setting. Therefore, there is adistinct need for appropriate and effective, non-toxic local deliverysystems for non-steroidal anti-inflammatory drugs.

Another approach to adhesion prevention which has met with a certaindegree of success entails the use of hyaluronic acid. Hyaluronic acidhas been tested in several different model systems to determine itsefficacy in preventing adhesion formation and reformation. There isextensive literature on the use of HA solutions to reduce postoperativeadhesion formation following abdominal and orthopedic surgery. Althoughthe results of various studies have been somewhat contradictory, thereappears to be a consensus that to achieve significant biologicalefficacy, HA compositions must be of high viscosity, high HAconcentration, and must contain high molecular weight and highlyconformationally ordered HA molecules (Balazs, U.S. Pat. No. 4,141,973).However, these properties limit the practical use of such HA solutions,especially in methods requiring injection of the compositions into smallspaces (e.g., tendon sheaths, joints) because, generally, solutionscontaining high concentrations of HA having a molecular weight greaterthan 8×10⁵ Daltons are not suitable for use as injectable formulations.

In a recent study, Abe et al. (J. Surg. Res. 55:451, 1990) evaluated theefficacy of sodium tolmetin in a medium of hyaluronic acid and phosphatebuffered saline (PBS) in the standard adhesion model involving theinjury of abrasion and devascularization of both uterine horns ofrabbits. Their treatment consisted of 15 ml of hyaluronic acid (2.5%,8000 CPS) and tolmetin sodium (1 mg/ml) in PBS, administeredintra-peritoneally via a syringe. Gross observations indicate that theoccurrence of mild to severe adhesions within 72-96 hours after surgeryoccurred in 25% of those animals treated with HA-tolmetin sodium,whereas mild to severe adhesions were observed in 60% of untreatedcontrols during the same time period.

In summary, developing appropriate and effective therapies forinhibiting inflammation and adhesion formation is still a majorchallenge. While NSAIDs have been shown to decrease the tissueinflammatory response and enhance fibrinolytic potential in peritonealtissues previous studies have shown that these drugs need to bedelivered to specifically targeted areas for several days to beeffective. In turn, while hyaluronic acid has demonstrated potential asa drug carrier, the concentrations and molecular weights required forbiological efficacy prohibit the practical use of this compound in aninjectable form. Therefore, more effective strategies for the treatmentof tissue trauma associated with adhesion formation are still needed.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that novel compositionscontaining high molecular weight hyaluronic acid (HA), non-steroidalanti-inflammatory drug(s) (NSAIDs), and an excess of at least oneorganic salt of at least one divalent metal (organometallic salts),unexpectedly form a composition with a viscosity that is easilyinjectable, but which increases in viscosity at the site of injection toform a gel thereby resulting in prolonged residence time of thecomposition in the biological environment. A study of such a compositionin an in vivo animal model indicates that these compositions demonstratea 100% incidence of reduced adhesion formation over control specimens,and thus, are more efficacious in preventing adhesions than previouslystudied formulations.

Therefore, in one aspect, the invention features injectable therapeuticcompositions containing hyaluronic acid, a non-steroidalanti-inflammatory drug, and at least one organic salt of at least onedivalent metal selected from the group consisting of magnesium acetate,zinc acetate, calcium gluconate and ferrous gluconate. Preferably, theorganic salt of the divalent metal is ferrous gluconate, which is alsocapable of acting as an anti-coagulant.

In preferred embodiments, the HA has a molecular weight of at least1.0×10⁶ Daltons; more preferably between 1.5×10⁶ and 3×10⁶ Daltons; evenmore preferably between 1.8×10⁶ and 2.6×10⁶ Daltons; and most preferablythe molecular weight of the HA is between 2×10⁶ and 2.5×10⁶ Daltons. Asused herein, the term "HA" means hyaluronic acid and any of itshyaluronate salts. Preferably, the HA used in the composition of theinvention is sodium-hyaluronate.

The non-steroidal anti-inflammatory drug preferably containsfree-carboxyl groups capable of interacting with the organic salt of adivalent metal. The NSAID can be in the form of a biologicallyacceptable salt (e.g., sodium), but preferably is in free acid form.Most preferably, the NSAID is chosen from group including tolmetin,ibuprofen, naproxen, peptides or mixtures thereof. Preferably, thepeptide is cyclosporin. NSAIDs, as is well known in the art. are definedas compounds which decrease prostaglandin synthesis but do not affectarachidonic acid levels.

The organic salts of the compositions preferably contain a fatty acidcomponent, e.g., acetic, gluconic and higher homologs thereof, and adivalent metal such as Ca²⁺, Mg²⁺, Zn⁺² or Mg⁺². Mixtures of organicsalts may also be used.

Also in preferred embodiments, the concentration of HA in thecomposition varies between 10 to 25 mg/ml, and is preferably is between15 to 20 mg/ml. The concentration of NSAID(s) is between a bout 0.5 to 3mg/ml, and is preferably 1 to 2 mg/ml. The organometallic saltconcentration is between 150-400 mg/ml, preferably between 150-250mg/ml, and more preferably between about 170-200 mg/ml.

The viscosity of the composition is greater than 10 dl/g, but less than18 dl/g, and more preferably is about 16 dl/g at 30° C. The ratio of HAto NSAID(s) is the composition is in the range between about 5 to 30mg/ml, preferably about 10 to 25 mg/ml, and more preferably is betweenabout 15 to 22 mg/ml. The ratio of the organic metal salt is in therange between about 15 to 4 mg/ml, preferably 12 to 6 mg/ml, and morepreferably about 10 to 8 mg/ml. In one preferred embodiment, thetherapeutic composition of the invention contains about 20 mg/mlhyaluronic acid, 200 mg/ml of divalent metal organic salt, and 1 mg/mlof cyclosporin A.

In another aspect, the invention features a method of inhibitinginflammation and adhesion formation in a mammal, preferably a human, byadministering to the mammal a therapeutic composition containinghyaluronic acid (HA), a non-steroidal anti-inflammatory drug (NSAID),and an organic salt of a divalent metal in a dosage effective to inhibitinflammation and adhesion formation.

In various embodiments of this aspect of the invention, the method isused for the treatment of a mammal at risk of adhesion formation due toa medical condition which is associated with inflammation and scarformation. By the term "medical condition" as used herein is meant anytrauma or disease of a bodily tissue which is associated with anincreased inflammation of the tissue and the formation of permanentfibrous adhesions. The terms "fibrous adhesions", "adhesion formation"and the like, as used herein, refer to dense, fibrous scar tissuecharacterized by a grade of 3-5 as defined by the grading scaledescribed by Rodgers et al. (1990, supra). Examples of diseasesassociated with the formation of fibrous adhesions include traumaticarthritis, osteoarthritis and bursitis. Examples of trauma include thosecaused by injury or surgical procedures.

In preferred embodiments, the method of the invention is used to treat amammal at risk of developing adhesions due to a surgical procedureincluding but not limited to peritoneal, pericardial, obstetric,gynecology, neurological, ophthalmic, orthoscopic, orthopedic, plastic,reconstructive, prosthetic, muscle or tendon. In especially preferredembodiments, the method involves inhibiting adhesion formation betweentendons and their sheaths during and after surgical procedures, e.g., infinger and knee joints.

In the method of the invention, the pharmaceutical composition of theinvention is preferably administered locally to the site at risk ofadhesion formation either during or alter trauma to the area (e.g.,during or after a surgical procedure). Preferably, the composition isadministered after the surgical procedure has been completed. Inpreferred embodiments, the method involves injection of the therapeuticcomposition using a 27-18 gauge needle, and more preferably using a23-20 gauge needle. "Local administration," as used herein, refers tothe contacting of a therapeutic composition in the immediate proximityof the tissue in the body where its therapeutic effect is desired.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although any method andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated by reference. Unless mentioned otherwise, the techniquesemployed or contemplated herein are standard methodologies well known toone of ordinary skill in the art. The materials, methods and examplesare illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph representing the correlation between gross evaluationgrades and gel composition.

FIG. 2 is a graph that illustrates the relationship between gelcomposition, inherent viscosity and in vivo effectiveness.

DETAILED DESCRIPTION OF THE INVENTION

Purified HA suitable for use in the therapeutic compositions of theinvention can be obtained from a number of commercial sources (GenzymeCorp., Cambridge, Mass.) or prepared according to methods described inthe art (See, for example, Balazs, U.S. Pat. No. 4,141,973). Highmolecular weight HA, i.e., greater than 1×10⁶ Daltons is best suited forthe compositions described herein. Molecular weights of HA can bedetermined by light scattering measurements as described in Yu et al.,"Rheological Characteristics of Microbially Derived Sodium Hyaluronate",American Chemical Society Proceedings Series--Harnessing Biotechnologyfor the 21^(st) Century, M. R. Ladisch and R. Bose eds., pp. 80-84,1992.

NSAIDs and organometallic salts appropriate for use in the therapeuticcompositions of the invention are also available from a number ofcommercial sources known to those skilled in the art and describedherein.

The major therapeutic components of the compositions of the invention,the HA, NSAIDs and organometallic salt can be combined as describedherein or by methods well known to the skilled artisan. Typically, theorganometallic salt will be in a concentration such that the ioniccomposition of the salt exceeds the free carboxyl groups of the HA anddrug. The concentration of the drug will depend on the condition to betreated, but will typically be in the range found to be effective forlocal, continuous delivery systems (e.g., see Rodgers, 1990, supra). Theparticular dosage of NSAID used in the composition will be influenced bya number of factors including the type and extent of the trauma, andoverall health and status of the particular patient. For mostapplications, the dose of the drug will range between 0.5 to 3 grams permilliliter of the formulation.

The amount of HA used in the formulation will generally be in an amountwhich produces a formulation with an initial viscosity outside thebiological environment that is greater than 10 dl/g at 30° C., but canbe optimized to suit the particular application. Viscosity of thepharmaceutical composition of the invention can be determined asdescribed herein, or by methods well known is by use of a Brookfieldcone and plate viscometer using the lowest shear rate that yield resultsgreater than 10% full scale.

Compositions for administration according to the method of the inventioncomprise a combination of HA, NSAID and an organic salt of a divalentmetal dissolved or dispersed in an acceptable carrier, preferably anaqueous carrier. A variety of aqueous carriers may be used, e.g., water,buffered 0.9% saline, and the like. The compositions may also containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like.

Injection of the therapeutic compositions according to the method of theinvention can be accomplished by means generally known to those skilledin the art. Generally, the amount of the therapeutic composition usedwill depend on the specific site of the body to be treated. For someapplications a single administration will often be sufficient to inhibitinflammation and adhesion formation at the desired site. However, wherecontinued or chronic pain is experienced (e.g., in joint inflammation),repeated applications may be used without adverse effect. Localadministration is preferred via a syringe according to well establishedtechniques, e.g., using a needle having a gauge size capable ofeffectively extruding the formulation while minimizing the invasivenessof the procedure.

Generally, surgical procedures, according to the method of the inventionemploy proper techniques for minimizing the risk of adhesion prevention(see, for example, Stangel et al. 1984. supra). The basic keys to goodsurgical technique include limiting trauma, ischemia, foreign bodyinteractions, hemorrhage, raw surfaces and infection. These techniquesare applicable to all types of surgical procedures and theysignificantly increase the potential for successful healing andrecovery.

Surgical trauma can be limited by handling tissues as little as possibleand using appropriate instrumentation. Ischemia, the most potentstimulus in adhesion formation, should be avoided by maintaining thevasculature of the tissue. Foreign bodies such as glove powder, lint,gauze particles, and suture remnants should be carefully removed fromthe surgical field. These various particles will initiate granulomatousreactions and trigger adhesion formation if left behind in vivo(Levinson and Swolin, Clin. Obstet. Gynecol. 23:1212, 1980).

It is also important to maintain hemostasis, thereby limiting the amountof blood at the site and decreasing one of the main sources of fibrin.Irrigation should be used as opposed to swabbing with gauze to preventabrasion of the tissue surfaces and the resultant cell damage. Finally,aseptic techniques and prophylactic antibiotic use are important inreducing the risk of infection post-surgically and limitingcomplications that can lead to adhesion formation.

EXAMPLE

In the following study, compositions which contained sodium hyaluronategels and different combinations of tolmetin sodium, naproxen sodium, andcalcium acetate were evaluated in a tendon adhesion model. Tolmetinsodium and naproxen sodium were selected based on their reportedeffectiveness in reducing inflammation in previous studies. Calciumacetate was chosen as a potential ionic cross-linking agent based on theinitial hypothesis that the calcium ions would preferentially exchangewith two sodium ions in the HA chains or the drug molecules in solution,thereby creating an ionic crosslink between the drugs and HA or betweenthe HA molecules.

Materials and Methods

Implants

Five different gel compositions were designed to study the individualeffects of hyaluronic acid (HA) (Genzyme Corp., Cambridge, Mass.),tolmetin sodium (R. W. Johnson Pharmaceutical Research Institute, SpringHouse, Pa.), naproxen sodium (Sigma Chemical Co., St. Lois, Mo.), andcalcium acetate (J. T. Baker, Inc., Phillipsburg, N.J.) on adhesionformation. All of the sodium hyaluronate used in the experimental studywas obtained from Lot C3040 (Genzyme Corp.), and had a molecular weightof 2.3×10⁶ Dalton. A base concentration of 2% w/v HA in phosphatebuffered saline (H=7.44) was used for each gel. Both the tolmetin sodiumand naproxen sodium were dosed in concentrations of 1 mg/ml. While thechosen concentration of calcium acetate was 0.216 g/ml. The individualcomposition of each gel was as follows:

1. HA: 20 mg/ml;

2. HA: 20 mg/ml, tolmetin sodium 1 mg/ml;

3. HA: 20 mg/ml, naproxen sodium 1 mg/ml;

4. HA: 20 mg/ml, calcium acetate: 0.216 g/ml; and

5. HA: 20 mg/ml, naproxen sodium 1 mg/ml, calcium acetate: 0.216 g/ml.

This concentration of calcium acetate was calculated to allow formaximum ionic cross-linking between the carboxyl groups of the HAbackbone and those in naproxen sodium. This concentration is alsoreasonably within the solubility range of calcium acetate.

Because hyaluronic acid is highly susceptible to bacterial degradation,aseptic techniques were used throughout the gel preparation phase ofthis study. All work with HA was conducted under a laminar flow hood(Labcona Purifier Class 11 Safety Cabinet, Labcona Corp., Kansas City,Mo.) which was regularly sterilized with an internal UV light source. AMettler AE 100 analytical balance (Mettler Instrument Corp., Highstown,N.J.) was thoroughly cleaned with 70% ethanol wipes and moved under thehood. It was then leveled and balanced, and exposed to UV light for 24hours. This scale was used to measure the components of each gel.

Measuring spatulas, magnetic stirbars, and 20 ml disposablescintillation vials (Kimble Glass, Inc., Vineland, N.J.) were placed insterilization bags (Baxter Healthcare Corp., Deerfield, Ill.) and weresteam sterilized in an autoclave (American Sterilizer, Erie, Pa.). Allglassware that was used was acid washed, and every article wasthoroughly wiped with 70% ethanol before entering the hood.

Twenty 0.200 gram aliquots of HA were measured and stored inscintillation vials at 4° C. A stock solution of phosphate bufferedsaline (PBS, ph=7.44) was prepared and filter sterilized using adisposable bottle top filter with a 0.22 μm cellulose acetate membrane(Corning Glass Workings, Corning, N.Y.). Two separate stock solutions oftolmetin sodium (1 mg/ml) and naproxen sodium (1 mg/ml) in PBS were madeand filter sterilized as described above. A fourth stock solution ofnaproxen sodium (1 mg/ml) and calcium acetate (0.216 g/ml) in PBS wasalso prepared. All solutions were stored at 4° C.

Gels were made up to 24 hours prior to surgical use by adding 10 ml ofthe appropriate stock solution to one aliquot of HA. A sterile magneticstirbar was added to each vial and the vials were sealed prior toremoval from the sterilization hood. Following manual agitation, eachgel as sonicated (Branson 1200, Branson Cleaning Co., Shelton, Conn.)for 1 minute. The vials were then placed on a Corning Stirrer/Hotplate(Corning Glass Works, Corning, N.Y.) and stirred on the "high-scale" for4 hours at room temperature. Following homogenous mixing, the gels wereonce again stored until 4° C. until surgery.

Viscosity Measurements

Solutions were prepared with a concentration of 0.1% w/v HA and theappropriate dilutions of the other components for solution viscositytesting. Initial tests were run in a 3° C. constant temperature waterbath, using a 100 capillary viscometer (Ostawald type). Fivemeasurements were taken for each individual solution to determine theinherent viscosity (n_(inh)). The solutions were then stored at 4° C.for 24 hours.

A second set of viscosity measurements were recorded 24 hours aftersolution preparation to determine the effect of storage prior tosurgery. These tests were also run at a constant temperature of 30° C.,with five measurements recorded for each solution.

Animals

The white leghorn chicken was chosen as the animal model. The anatomicalarrangement of tendons in the foot of the chicken is very similar tothat of the human hand (Craver et al., Surg. 64(2):437, 1968) makingthis species clinically relevant. The surgical model we have adopted isbased on one designated by Daley et al. (Proc. Orthop. Res. Soc. Ann.Mtg. 38:1-3, 1992) for the specific application of testing biomaterialsfor adhesion prevention.

Chickens were also chosen due to their vulnerability, costeffectiveness, and ease of maintenance. Fifty-six female, adult, whiteleghorns were obtained from Morgan Poultry Farm (Clemson University,Clemson, S.C.). They were housed two birds per cage by Clemson ResearchServices at either Jordon Hall or the Poole Agriculture and SciencesBuilding for one week prior to surgery. The birds weighed between 1.03kg and 2.02 kg per-operatively and between 0.92 and 2.24 kg at necropsy.

Surgical Protocol

Each animal was given Buprenorphine (0.03 mg/kg, IM) up to 2 hoursbefore surgery as a premedication. Anesthesia was induced with 2-3%Forane™ in oxygen (2 l/min) administered through a fitted mask. Onceinduced, the birds were intubated with a 3 mm ID endotracheal tube andmaintained using 2-3% Forane™ in oxygen (2 l/min). Respiratory rate andcapillary refill time were used to monitor anesthetic depth and oxygenperfusion.

A major concern in avian surgery is body heat loss. Therefore all birdswere placed on a water blanket peri-operatively and wrapped in towelspost-operatively. Also, the operative foot was prepped with alternatingsolutions of Nolvasan Surgical Scrub (Aveco, Co., Inc., Fort Dodge,Iowa) and sterile 0.9% Sodium Chloride Irrigation USP (Baxter HealthcareCorp., Deerfield, Ill.), to prevent evaporation cooling that oftenoccurs with alcohol.

(a) Pilot Study

Under tourniquet control, a 2.5 cm incision was made in the plantaraspect of the long central digit of the chicken's left foot. The tendonsheath, or peritenon, was incised exposing the flexor sublimis tendon.The sublimis was retraced along with the peritenon to expose the flexorprofundus tendon. A 50% tenotomy was made in the flexor profundus andsutured with a modified Kesler stitch using 3-0 Ethibond™ suture(Ethicon, Inc., Somerville, N.J.).

The peritenon was closed with a simple continuous stitch using 3-oEthibond™. A gauze dressing was applied to the foot covering theincision. Padding and a fiberglass cast (Carapace, Inc., Tulsa, Okla.)were applied with the toes in hyperextension and the foot in flexion.This type of immobilization relieves tension at the repair site in thetendon and allows the bird to walk easily. Co-Flex wrap (Andover CoatedProducts, Inc., Marblehead, Mass.) was applied to each cast to minimizecontamination, and this wrap was changed regularly.

(b) Experimental Study

In order to test the effectiveness of each gel, animals were randomlyassigned to one of six groups, with eight animals per group. One groupserved as the control, while the other five groups each received adifferent gel treatment.

The surgical procedure was slightly modified for the experimental studyby changing the sutures that were used. The flexor profundus tendon andthe tendon sheath were sutured with 3-0 Chromic (Ethicon, Inc.,Somerville, N.H.). Following closure of the sheath, 0.2 ml of each gelwas delivered to the intra-sheath region by a tuberculin syringe. Theskin was closed with 3-0 Silk (Ethicon, Inc., Somerville, N.H.). Allother aspects of the surgical procedure remained the same throughout thein vivo study. There were no notable modifications or complications inany aspect of anesthesia or surgery. All animals maintained respiratoryrates and anesthetic depths within normal ranges. The surgical procedurelasted approximately thirty to forty minutes per animal.

Recovery from inhalation of anesthesia was quite rapid in birds(approximately 3-5 minutes). Initial recovery from anesthesia was rapidand without complication for all of the animals in the study. Duringthis time period each animal was given 15 cc of Lactated Ringer'sInjection USP (McGaw, Inc., Irvine, Calif.) subcutaneously to maintainproper hydration. Each bird was alert and on its feet within ten tofifteen minutes. In general, the chickens exhibited normal behavior fromimmediately following surgery until euthanasia two weeks later. Thisincluded regular eating habits, alert and active behavior, full weightbearing on each foot, no pronounced limping, no need for additionalanalgesics, and no significant loss of body weight (<<20% of pro-surgeryweight).

All birds were returned to their pre-operative housing within a fewhours of surgery. Body weight, appetite, behavior and weight bearing onthe casted foot was closely monitored to assess post-operative health.Buprenorphine (0.03 mg/kg, IM) was given 12 hours post-operatively andsubsequently every eight hours if needed for pain. A humane endpoint wasset at 20% loss of body weight.

Four of the fifty-six animals had to be removed from the study due topost-operative complications. Three of these birds were euthanized priorto the two week time period. Each one developed a pronounced limp withinseveral days of surgery. They were treated with Buprenex (0.03 mg/kg,IM, every eight hours) but did not respond to the analgesic treatment.These birds were lethargic and reluctant to bear weight on theirimmobilized foot. Additionally, they had depressed eating habitsaccompanied by significant weight loss. None of these birds reached thehumane endpoint of 20% body weight loss; however, they were euthanizeddue to signs of unalleviated pain.

Upon examination, these three specimens did not show any gross signs ofinfection. There was no evidence of swelling, redness, exudate, or poorhealing. There were some signs of light bruising on the distal ends ofseveral of the toes involved, possibly caused by the cast itself. If thecast was applied too tightly, it may have caused bruising anddiscomfort. This is a plausible explanation for the limping observed inthese birds. It should also be noted that each of the affected animalscame from different experimental groups showing no bias toward aspecific implant.

The fourth bird was not prematurely euthanized; however, the flexorprofundus tendon was completely ruptured at the suture site when thegross evaluation was performed. This was not consistent with thesurgical model and was therefore eliminated. The cause of this ruptureis unknown.

Euthanasia and Specimen Retrieval

After 2-4 weeks for the animals in the pilot study, and 2 weeks forthose in the experimental study, each chicken was terminated with carbondioxide in a pre-charged chamber. The left foot of each bird was removedabove the cast and the casts were cut using a Richard Oscillating Saw(Richards Manufacturing Co., Memphis, Tenn.). Casts, padding, and gauzewere discarded and the feet were labeled and stored below 0° C. untilgross evaluation could be conducted.

X-Ray Analysis

A radio-opaque gel of 2% w/v HA was formed in 50% PBS and 50% Hypaquesodium (Sanofi Winthrop Pharmaceuticals, New York, N.Y.). Initial x-rayswere taken of a cadaver foot as a baseline for comparison. The gel wasthen applied to four cadaver feet using the described surgicalprocedures. Anteroposterior and mediolateral x-rays were taken of eachfoot to verify the placement of the gel during its surgical application.

Gross Evaluation

All specimens were grossly dissected in groups of at least twelve forcomparative purposes. The feet were thawed and observations ofinflammation, and the skin closure were noted. Skin sutures were removedand the toe was dissected through the original incision. Subcutaneousinflammation and adhesion formation was recorded. The peritenon gradingscale (Table I) was used to evaluate the extent and severity of adhesionformation within the intra-sheath region.

                  TABLE I                                                         ______________________________________                                        Qualitative Grading Scale.sup.a                                               Grade         Evaluation                                                      ______________________________________                                        1             No Adhesion                                                     2             Filmy (separable)                                               3             Mild (not separable)                                            4             Moderate (35-60% of area)                                       5             Severe (greater than 60% of area)                               ______________________________________                                         .sup.a (Rodgers et al., 1990 supra).                                     

More specifically, for the gross evaluation all specimens werecategorized based on the maximum severity of the adhesions present. Forexample, if a particular specimen exhibited primarily thin, filmyadhesions but had one small band of non-separable fibrous tissue, it wasclassified as a grade 3 rather than grade 2, its predominant type.

Each grade division on the scale was associated with a set of specificgross characteristics. Grade 5 specimens were characterized by dense,fibrous adhesions covering virtually the entire surgical field. Theyinvolved the profundus and sublimis tendons, along with the tendonsheath and underlying bone. There were adhesions present between thetendons and form the tendons to the surrounding structures.

Grade 4 specimens were similar to those in grade 5; however, thescarring was slightly less extensive. The adhesions were stillcharacterized by dense, fibrous scar tissue but there was lessinvolvement at the profundus tendon. Adhesions were most predominantbetween the flexor sublimis and the tendon sheath and between the twotendons.

The specimens that were classified as grade 3 were quite different fromthe previous two groups. These adhesions were very predominant betweenthe tendon sheath and the flexor sublimis. In some cases these twotissue structures were firmly adherent throughout the length of thesurgical field. In other cases the majority of the adhesions between thesheath and sublimis were filmy with the presence of non-separablefibrous bands in one or two localized areas. There was almost noinvolvement of the flexor profundus tendon. Any adhesions to this tendonwere minimal and filmy in nature.

Grade 2 adhesions are filmy and easily separable as defined by thegrading scale (Rodgers et al. 1990, supra). These were easilydistinguishable from the more severe grades 3-5 because there were nofibrous bands of scar tissue present between adjacent tissue structures.Grade 2 specimens were characterized by thin, wispy adhesions presentbetween the tendon sheath and the flexor sublimis. There was virtuallyno involvement of the flexor profundus and this tendon was able to glidefreely in both directions upon manipulation. The adhesions that werepresent between the sheath and the sublimis were easily dissectable andthe tissue structures separated from each other with very little appliedforce.

A grade of 1 in the chosen scale is representative of no adhesionformation. Even under ideal surgical conditions, there will be somedegree of scar tissue formation. Scarring was visible in thesubcutaneous tissue of all specimens; however, it was proportional tothe overall quality of healing and adhesion formation. Ideally,specimens that are classified as grade 1 should appear identical totheir pre-operative condition. This was not the case with any of theanimals evaluated in this study. However, a number of specimens werejudged to be significantly different from the typical grade 2 category.These specimens showed a very limited incidence of extremely filmyadhesions between the sheath and flexor sublimis. The tissue structuresseparated immediately upon dissection of the tendon sheath. Ultimately,these specimens were assigned a grade of 1.5 for the purpose of dataevaluation. While they did not meet the stringent criteria establishedfor grade 1, they were significantly different from grade 2 adhesions intheir extent and quality.

Following gross evaluation, specimens were disarticulated at themetacarpophalangeal joint and placed in containers of 10% neutralbuffered formalin for histological preparation.

Histological Preparation

Fourteen specimens were selected and submitted for histologicalprocessing and evaluation. They were chosen based on their grossevaluation grades and their experimental groups (Table II). Specimenswere trimmed grossly of any excess bone, skin, etc. and placed in tissuecassettes. They were processed in the Tissue Tek V.I.P.™ TissueProcessor (Miles Scientific, Miles, Inc., Mishawaka, Ind.) using amodified version of program zero (Table III). following processing thespecimens were removed from the tissue processor and blotted dried toremove excess xylene.

                  TABLE II                                                        ______________________________________                                        Specimens Submitted for Histological Evaluation                               Specimen No.  Test Group.sup.a                                                                          Gross Grade                                         ______________________________________                                        94-C-51       CONTROL     5                                                   94-C-53       CONTROL     4                                                   94-C-56       CONTROL     5                                                   94-C-61       HA          4                                                   94-C-65       HA          3                                                   94-C-70       HA + TS     3                                                   94-C-71       HA + TS     2                                                   94-C-75       HA + NS     2                                                   94-C-78       HA + NS     3                                                   94-C-80       HA + NS     1.5                                                 94-C-82       HA + CA     3                                                   94-C-92       HA + CA     2                                                   94-C-93       HA + CA + NS                                                                              2                                                   94-C-96       HA + CA + NS                                                                              1.5                                                 ______________________________________                                         .sup.a ts: tolmetin sodium, NS: naproxen sodium, CA: calcium acetate     

                  TABLE III                                                       ______________________________________                                        Modified Program Zero                                                         Solution.sup.a                                                                           Time        P/V.sup.b,c                                                                           Temperature                                    ______________________________________                                        10% NBF    20 min.     Y       40° C.                                  10% NBF    1 hr.       Y       40° C.                                  70% EtOH   30 min.     Y       40° C.                                  80% EtOH   30 min.     Y       40° C.                                  95% EtOH   45 min.     Y       40° C.                                  95% EtOH   45 min.     Y       40° C.                                  100% EtOH  45 min.     Y       40° C.                                  100% EtOH  45 min.     Y       40° C.                                  Hemo-De    40 min.     Y       35° C.                                  Xylene     1 hr.       N       35° C.                                  ______________________________________                                         .sup.a EtOH: ethanol; .sup.b P/v: Pressure/Vacuum; .sup.c Y=yes, N=no    

The selected specimens were infiltrated in a series of graded solutionsof TECHNOVIT 7100 (Delaware Diamond Knives, Wilmington, Del.), aglycomethacrylate embedding medium. All diluted solutions were mixedwith 100% ethanol to yield the appropriate concentrations. The tissuecassettes were placed in 50% TECHNOVIT 7100 for 48 hours. They were thentransferred to 75% TECHNOVIT 7100 for another 48 hours. Finally, thecassettes were infiltrated in 100% TECHNOVIT 7100 for 72 hours. All ofthe infiltration mediums were agitated under a vacuum and protected fromlight.

Once infiltrated, each specimen was embedded in a solution of 100%TECHNOVIT 7100 and Hardener II, mixed in a 15:5 ratio, as specified forlarge specimens in the package insert. Specimens were arranged indisposable plastic molds (4.5 cm×2.7 cm×2.0 cm) and covered with 15 mlof the embedding solution. They were placed in a desiccator andpolymerized under vacuum for 24 hours.

Following polymerization, the tissue blocks were very rubbery. Normallythe polymerized blocks are a hard, solid mass. These blocks were not ofthe appropriate hardness to cut on the Polycut E Microtome(Reichert-Jung, Reichert Scientific Instruments, Buffalo, N.Y.). Theblocks were subjected to heat, cold, and vacuum conditions in attemptsfor further polymerization. These unsuccessful attempts werediscontinued 48 hours later, and specimens were recovered andre-embedded.

Slides were stained with Hematoxylin and Eosin, Masson's Trichrome (PolyScientific Corp., Bay Shore, N.Y.), and a modified Trichrome for GMAembedded tissues (Osheroff and Ruffing, J. Histotech. 8:92, 1985). Thespecific staining procedures are listed in appendix a.

Histological Evaluation

The histological evaluation was done subjectively and qualitatively.Observations focused on the amount of granulation tissue present, thepresence of inflammatory cells, the relative thickness of granulationtissue and adhesion formation around the tendons, and location ofpost-surgical adhesions. Due to the difficulty in tissue processing, thequality of slides was not sufficient to perform a detailed histologicalanalysis. However, general observations were made and correlated withthe results of the gross evaluation.

Statistical Analysis

Microsoft Excel (version 4.0a) was used to perform analysis of variance(ANOVA) and student t-tests to evaluate the results of the gel viscositytests and the in vivo gross evaluation. P-values less than 0.05 wereconsidered significant differences, while p-values less than 0.01 wereconsidered highly significant.

Results and Discussion

Gel Characterization

All of the gels were prepared as described above without modification.Solution viscosity tests were used to calculate inherent viscosity(η_(inh)), verify the stability of the gel compositions during storage,and establish a relationship between gel viscosity and effectiveness invivo. Hyaluronic acid is an absorbable polymer susceptible to bacterialdegradation. Degradation of the polymer chains results in a decrease inmolecular weight. The inherent viscosity of a polymer is proportional toits molecular weight. Thus, significant degradation of the polymer wouldresult in a substantial decrease in inherent viscosity.

The inherent viscosity of each gel was determined immediately followingpreparation (t=0 hr) and twenty-four hours later (t=24 hr) to determinethe effect of storage at 4° C. None of the gels were stored for morethan 24 hours prior to surgical implantation, therefore their stabilitywas not investigated beyond this time period. The results of thesolution viscosity tests arc listed in Table IV. All data is shown asinherent viscosity in the units of deciliters per gram (dl/g). Each datapoint is the average of five repetitions of one solution.

                                      TABLE IV                                    __________________________________________________________________________    Inherent Viscosity Results                                                          HA HA + NS                                                                             HA + TS                                                                            HA + CA                                                                             HA + CA + NS                                                                          Average                                     Time (hr)                                                                           (dl/g)                                                                           (dl/g)                                                                              (dl/g)                                                                             (dl/g)                                                                              (dl/g)  (dl/g)                                      __________________________________________________________________________     0    18.91                                                                            19.50 19.48                                                                              16.10 16.21   18.04                                       24    18.74                                                                            19.64 19.58                                                                              16.13 16.05   18.02                                       __________________________________________________________________________

Although there are slight fluctuations in the inherent viscosities ofthe different gels at 0 and 24 hours, none of these differences arestatistically significant. There is no specific trend in the data.Analysis of variance (ANOVA) does not indicate any significantdifference in the overall average η_(inh) between the two time periods(p+0.955). These results confirm the stability of each gel compositionduring storage prior to surgery.

Necropsy

In general, the tissues of the involved feet appeared healthy andnormal. The skin incisions were completely healed and there were nogross signs of infection. The necropsy results indicated that all of theimplanted materials were sufficiently sterile.

x-Ray Analysis

Samples of radio-opaque HA gel were injected into four different cadaverfeet using the surgical procedure described for the experimental study.X-rays of the feet showed that the gels are concentrated and confined toa localized space between the distal half of the proximal phalanx andthe proximal half of the medial phalanx. This is consistent with thelocation of the surgical field. Mediolateral views of the specimensillustrate the presence of gel in the soft tissues of the toe. Thisconfirms that the implants were properly delivered to the targeted areain this surgical model.

Gross Evaluation

The in vivo research involved a pilot study and a subsequentexperimental study to evaluate the effectiveness of each gelcomposition. The pilot study was primarily used to become familiar withthe surgical model and to evaluate the consistency of results. Thegrades of from gross evaluation of the animals in the initial pilotstudy ranged from 2 to 4, with little variation between the two and fourweek time period. These results were judged to be insufficient in termsof severity for the control portion of the study.

The objective was to elicit a response in the 4 to 5 range from thesurgical model alone. The sutures used in the pilot study were changedfrom Ethibond™ to Chromic and Silk in order to produce a more severeresponse. This was a successful modification and the control group had amean adhesion score of 4.875/5. All of the grade 5 adhesions reportedthroughout the study were confined to the control group where theyrepresented 87.5% of the data of this set. Based on these results, theanimal model was judged to be both effective and consistent in producingsevere tendon adhesions. Its effectiveness can be attributed to thecombination of tendon sheath incisions, tendon injury and suturing, andfull immobilization (Matthews and Richards, J. Bone and Joint Surg.58-b(2):230, 1976).

The results from the experimental evaluation of each gel composition areshown in FIG. 1. This graph represents the data as a 95% confidenceinterval about the mean for each data set. ANOVA was performed on thedata with the null hypothesis that all of the group means were equal.This hypothesis was rejected and the data was further evaluated forspecific differences.

Results from the statistical analysis strongly supported the fact thatall of the gel treatments were significantly effective in limitingadhesion formation as compared to the control group. The leastsignificant difference was between the simple HA gel and the control,which yielded a p-value much less than 0.01. In addition, all medicatedor "cross-linked" treatments (those with NSAIDs and/or calcium acetateadded) were significantly more effective than the HA alone (Leastsignificant difference: p<0.05). There was no statistical differencebetween the effectiveness of the NSAIDs tolmetin sodium and naproxensodium.

The addition of calcium acetate to the HA gel significantly improved itseffectiveness (p<0.01). Calcium acetate also had a significant positiveeffect when it was added to the HA-naproxen sodium gel. The mean scoreof the HA+CA+NS gel was significantly lower than both the HA+NS score(p<0.05) and the HA+CA score (p<0.05).

It is apparent that the addition of NSAIDS and calcium acetate, as a"cross-linking" agent, has a profound effect on the in vivo performanceof the gels in this animal model. It was hypothesized that this effectwould be partially due to the anti-inflammatory properties of the twodrugs and also to the expected increase in viscosity from cross-linkingthe gels. However, the inherent viscosities of the gels with the calciumacetate were substantially lower than expected prior to theiradministration. There was actually a decrease in η_(inh) between thesimple HA and HA-NSAID gels and those gels crosslinked with calciumacetate.

Hyaluronic acid has a high inherent viscosity because it is an extremelylarge molecule (MW: 2.3×10⁶ Dalton). The increase in inherent viscositybetween the HA gels and the HA-NSAID gels is due to the incorporation ofthe relatively small drug molecules in the HA gel matrix, although thereis no stimulus for chemical interaction between these molecules. Crosslinking increases the molecular weight of the polymer, and thus wasexpected to increase the viscosity. However, unexpectedly,"cross-linking" with calcium acetate did not produce an initial increasein viscosity, but rather resulted in a lowering of the viscosity of thecomposition containing all three components.

FIG. 2 illustrates the relationship between gel composition, inherentviscosity, and in vivo effectiveness. Hypothetically, gels containingthe combination of HA and NSAIDS with the highest viscosity shoulddemonstrate the optimal in vivo performance. In contrast to the expectedresult, the HA+CA+NS gel, which demonstrated a significantly lowerviscosity was the most effective in preventing adhesion formation.

One explanation for this unexpected observation of the relationshipbetween inherent viscosity and efficacy is that the addition of calciumacetate in a closed system such as a preparation vial or an Oswaldviscometer does not affect the molecular volume of the hyaluronic acid,but rather changes the ionic concentration of the gel resulting in anoverall lower inherent viscosity. When these gels are then placed in anopen system, such as the in vivo environment, ion exchange occurs andthe byproduct of this reaction rapidly diffuses out of the hyaluronicacid matrix. In this case, the reaction byproduct is sodium acetate, andthis in situ ionic cross linking leaves a higher viscosity gel at thesurgical site. This would account for the effectiveness of the HA+CA andHA+CA+NS systems. The increased viscosity upon application yields alonger in vivo residence time for these compositions.

Histological Evaluation

The quality of the histological slides was only average due to thedifficulties encountered in processing and embedding the tissuespecimens. The procedure that was used to salvage and re-embedded thetissues resulted in blocks of appropriate harness for cutting on themicrotome. However, when sections were cut from the blocks there was anotable difference in consistency of the polymeric embedding mediumwithin the tissue as opposed to outside the tissue. Consequently, thetissues did not cut as well as expected. The medium was too soft toprevent shattering of the bone although this did not affect theevaluation. There was a significant amount of tearing and wrinkling intissue sections. The longitudinal sections were most adversely affectedbecause of the larger tissue surface area involved in cutting thesections.

Another difficulty in the histological evaluation was encountered instaining the slides. Hematoxylin and eosin stains were successful;however, the Masson's trichrome and the modified trichrome proceduresyielded uneven staining patterns. Trichrome normally stains nucleiblack, and cytoplasm, keratin, muscle fibers and intracellular fibersred; and collagen blue. Both of the procedures used in his studyresulted in an uneven, somewhat random distribution of red a blue stain.This complication made it impossible to evaluate these slides in termsof collagen production. Therefore, and overall qualitative approach wastaken in evaluating the histological slides. There were no notabledifferences in the characteristics of specimens from different treatmentgroups that were assigned to the same gross grade. Observations werethus made based on the amount of tissue in the intr-sheath space, thepresence and location of adhesions, and the overall cellular response.These observations were then correlated with the grades from the grossevaluation.

The intra-sheath space of grade 5 specimens was filled with fibrousconnective tissue indicative of dense adhesions. This tissue was verycellular, but notably avascular. Macrophages and fibroblasts were thepredominant cellular components; however, eosinophils. giant cells, andplasma cells were also present. Additionally, a thick fibrous tissuecapsule was observed around the circumference of each tendon. Adhesionswere present between the tendons and from the tendons to the surfaces ofthe sheath and the bone.

Grade 4 specimens also exhibited a tissue response indicative ofadhesion formation: however, to a lesser degree than grade 5 specimens.The presence of fibrous tissue was more predominant at the periphery ofthe tendons. Adhesions were present between the tendons and from thetendons to the sheath. The scar tissue present was less cellular incomparison to the grade 5 specimens, with macrophages and fibroblastsbeing the predominant cell type.

The amount of fibrous scar tissue decreased with each improvement ingross grades. Grade 3 specimens displayed little evidence of fibrousscar tissue around the profundus tendon; however, there were denseadhesions between the sublimis tendon and the sheath. Grade 2 specimensshowed scant evidence of fibrous tissue around the profundus and a fewareas of limited contact between the sheath and the sublimis. There wereno signs of chronic inflammatory cells in any of these specimens.Unfortunately, the grade 1.5 specimens were longitudinal sections andtheir poor quality made it impossible to evaluate their histologicalcharacteristics.

Clinical Relevance

Ultimately the goal in post-surgical tendon adhesion prevention is torestore the functional performance of the affected digit. Dense,fibrous, non-separable adhesions restrict the gliding function of theflexor tendons. But filmy, separable adhesions are much more likely toundergo significant remodeling in the clinical setting with treatmentssuch as physical therapy. These types of adhesions (categorized as grade2 or less in this study) take much less force to break, and usuallynever present any significant functional impairment to the patient.

Therefore, it is interesting to relate this clinical perspective to theresults of this animal study. Exactly two-thirds of all of the treadedspecimens exhibited adhesions of grade 2 or less. The incidence ofadhesions less than or equal to grade 2 was 82.76% in all of themedicated and/or crosslinked treatment groups. Most surprisinglyhowever, 100% of the animals in the HA+CA+NS treatment group had scoresof 2 or less. The success of these materials in this animal study ofsevere trauma certainly indicates the efficacy of these combinations inclinical adhesion prevention.

Without being bound by any one theory, according to the results of thestudy described herein, the excess organometallic salt concentration inthe compositions of the invention appears to promote intr- and/orinter-molecular ion-binding of the HA molecules leading to substantialdecrease in the polymer hydrodynamic volume and, hence, viscosity. Onceinjection of the composition into the biological environment within aconstrained space occurs, there is an exchange of the metal. Ions withbody fluids which produces an increase in the viscosity of theformulation to a gel form, and thereby provides an initial high coverageof the traumatized space. Further, due to the low pKa value of thetypical NSAID, the solubility of the drug is decreased upon interactingwith the divalent ion. This leads to minimized initial drug burst and aprolonged release period. Thus, the compositions of the invention are asignificant improvement over those previously investigated became thebiological efficacy of the components are maximized.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. For example, the compositions of the method may also beuseful for the local delivery of other bioactive compounds includingpeptides and proteins, e.g., intravitreally.

What is claimed is:
 1. A therapeutic composition for use in inhibitingtissue inflammation and adhesion formation in a mammal, said compositioncomprising hyaluronic acid, a non-steroidal anti-inflammatory agent, andat least one organic salt of at least one divalent metal selected fromthe group consisting of magnesium actate, zinc acetate, calciumgluconate and ferrous gluconate.
 2. The therapeutic composition of claim1 wherein the molecular weight of the HA is at least 1.0×10⁶ Daltons. 3.The therapeutic composition of claim 1 wherein the non-steroidalanti-inflammatory agent is selected from the group consisting oftolmetin, ibuprofen, naproxen and peptides.
 4. The therapeuticcomposition of claim 1 wherein the non-steroidal anti-inflammatory agentis a peptide.
 5. The therapeutic composition of claim 4 wherein thepeptide is cyclosporin A.
 6. The therapeutic composition of claim 1wherein the organic salt of a divalent metal is magnesium acetate. 7.The therapeutic composition of claim 1 wherein the organic salt of adivalant metal is zinc acetate.
 8. The therapeutic composition of claim1 wherein the organic salt of a divalant metal is calcium gluconate. 9.The therapeutic composition of claim 1 wherein the organic salt of adivalant metal is ferrous gluconate.
 10. The therapeutic composition ofclaim 1 wherein the viscosity of the composition is less than 18 dl/g.11. A therapeutic composition for use in inhibiting tissue inflammationand adhesion formation in a mammal, said composition comprisinghyaluronic acid, cyclosporin A and at least one organic salt of at leastone divalent metal selected from the group consisting of magnesiumacetate, zinc acetate, calcium gluconate and ferrous gluconate.
 12. Thetherapeutic composition of claim 11 wherein the organic salt of adivalent metal is ferrous gluconate.
 13. A method of inhibiting ormediating tissue inflammation in mammal tissue, said method comprisingadministering to said mammal a therapeutic composition comprisinghyaluronic acid, a non-steroidal anti-inflammatory agent, and at leastone organic salt of at least one divalent metal in a dosage effective toinhibit or mediate local inflammation and adhesion formation in saidtissue, wherein said organic salt of a divalent metal is selected fromthe group consisting of magnesium acetate, zinc acetate, calciumgluconate and ferrous gluconate.
 14. The method of claim 13 wherein saidmammal is at risk of tissue adhesion formation as a result of tissueinjury.
 15. The method of claim 13 wherein said mammal is at risk oftissue adhesion formation which is pathological in origin or the resultof surgery.
 16. The method of claim 15 wherein said surgery occurs atthe site of a tendon.
 17. The method of claim 13 wherein saidadministration is local.
 18. The method of claim 17 wherein the localadministration is intraocular or at the site of the synovial fluid at anarticulating joint.
 19. The method of claim 18 wherein the non-steroidalanti-inflammatory agent is a peptide.
 20. The method of claim 19 whereinthe peptide is cyclosporin A.